Multi-Epitope Vaccines
It is known that B-cell epitopes, T-helper cell epitopes, and cytotoxic T lymphocytes epitopes all play important roles in these two immune responses. Obviously, broad spectrum and long lasting humoral and cellular responses should be induced for effective vaccination. There are still no broad spectrum and effective vaccines against viruses with high mutation rates, such as influenza virus and human immunodeficiency virus.
There is a close relationship between antigen dose and the efficiency of the specific B-cell response. Studies using a chemically coupled carrier protein and epitope peptide system, consisting of the same amount of carrier protein coupled with varying amounts of epitope peptide, have shown that epitope density dramatically affected T helper cell-dependent IgG responses (Jegerlehner et al., Eur J. Immunol. 2002, 32:3305-14). Liu et al. (Vaccine. 2004 23(3):366-71.) observed a positive effect of epitope density on the humoral response of mice and rabbits immunized with glutathione-S-transferase fusion proteins bearing various numbers of copies of the M2e peptide epitope (1, 2, 4, 8, and 16 copies) of the M2 protein of the influenza virus. In the same study, a lethal challenge assay showed that the fusion protein with the higher epitopes densities resulted in higher survival rates and slower weight losses.
Multi-epitope vaccines, namely vaccines comprising more than one epitope, have been developed for a wide variety of applications. A non-exhaustive list of examples includes, e.g., a recombinant multivalent vaccine for streptococcal bacteria disclosed in U.S. Pat. No. 6,063,386; a vaccine for treatment of malaria which comprises a single protein comprising peptides derived from different stages of the life cycle of the parasite Plasmodium falciparum, disclosed in U.S. Pat. No. 6,828,416; anti-tumor immunogenic compositions comprising a polypeptide comprising prostate stem cell antigen epitopes, disclosed in US Pat. Application 2007/0056315; and multi-epitope anti-viral vaccines against HIV (International Publication WO 01/24810), rubella virus (see International Publication WO 93/14206), and Hepatitis C virus (International Publication WO 01/21189).
International publication WO 2006/069262 discloses compositions, fusion proteins and polypeptides comprising Pathogen Associated Molecular Patterns (PAMP) and epitopes of influenza viral proteins used to stimulate immune responses in a subject. PAMPs are molecular motifs (e.g., proteins, peptides, nucleic acids, carbohydrates, lipids) found in microorganisms that can trigger an innate immune response in a host, i.e., act as adjuvant. In some embodiments the fusion proteins include multiple copies of the M2e influenza epitope. International publication WO 2006/078657 discloses similar fusion proteins and polypeptides comprising one or more PAMP and multiple epitopes of flaviviral proteins.
Influenza
Influenza is a disease caused by viruses of three main subtypes, Influenza A, B and C, which are classified according to their antigenic determinants. The influenza virion consists of a single stranded RNA genome closely associated with a nucleoprotein (NP) and enclosed by a lipoprotein envelope lined by matrix protein (M1) and carrying two major surface glycoprotein antigens, haemagglutinin (HA) and neuraminidase (NA). The HA and NA glycoproteins are most susceptible to change; for example, there are 16 immune classes of HA and 9 different NA classes that provide the basis for the different influenza virus subtypes like H1N1 or H3N2. Influenza A virus has an additional transmembrane glycoprotein, M2, which is highly conserved between the different HN subtypes. The M2 gene encodes a protein having 96-97-amino-acids that is expressed as a tetramer on the virion cell surface. It is composed of about 24 extracellular amino acids, about 19 transmembrane amino acids, and about 54 cytoplasmic residues (Lamb et al, Cell. 1985; 40:627-633.).
Influenza A and B viruses are the most common causes of influenza in man. Influenza has an enormous impact on public health with severe economic implications in addition to the devastating health problems, including morbidity and even mortality. Infection may be mild, moderate or severe, ranging from asymptomatic through mild upper respiratory infection and tracheobronchitis to a severe, occasionally lethal, viral pneumonia. Influenza viruses have two important immunological characteristics that present a challenge to vaccine preparation. The first concerns genetic changes that occur in the surface glycoproteins every few years, referred to as “antigenic drift”. This antigenic change produces viruses that elude resistance elicited by existing vaccines. The second characteristic of great public health concern is that influenza viruses, in particular influenza A virus can exchange genetic material and merge. This process, known as “antigenic shift”, results in new strains different from both parent viruses, which can be lethal pandemic strains.
Influenza Virus Antigens and Vaccine Production
Immunization towards influenza virus is limited by the antigenic variation of the virus and by the restriction of the infection to the respiratory mucous membranes. The influenza vaccines currently available are based either on whole inactive virus, on viral proteins presented on the surface of bacterial cells, or on flagellin bearing viral antigenic determinants. HA is a strong immunogen and is the most significant antigen in defining the serological specificity of the different virus strains.
The HA molecule (75-80 kD) comprises a plurality of antigenic determinants, several of which are in regions that undergo sequence changes in different strains (strain-specific determinants) and others in regions which are conserved in many HA molecules (common determinants). Due to these changes, flu vaccines need to be modified at least every few years.
Many influenza antigens, and vaccines prepared therefrom, are known in the art. U.S. Pat. No. 4,474,757 discloses a vaccine against influenza virus infections consisting of a synthetic peptide corresponding to an antigenic fragment of HA attached to a suitable macromolecular carrier, such as polymers of amino acids or tetanus toxoid.
PCT International Publication WO 93/20846 to some of the inventors of the present invention teaches a synthetic recombinant vaccine against a plurality of different influenza virus strains comprising at least one recombinant protein comprising the amino acid sequence of flagellin and at least one amino acid sequence of an epitope of influenza virus HA or NP, or an aggregate of said chimeric protein. Following this approach, a synthetic recombinant anti-influenza vaccine based on three epitopes was found to be highly efficient in mice. The exemplified vaccines included flagellin chimeras comprising the HA 91-108 epitope, a B-cell epitope from the HA which is conserved in all H3 strains and elicits anti-influenza neutralizing antibodies, together with one or both T-helper or CTL NP epitopes (NP 55-69 and NP 147-158, respectively), which induce MHC-restricted immune responses. A vaccine comprising a combination of the three above mentioned chimeras was considered to afford the best protection to viral infection.
U.S. Pat. No. 6,740,325 to some of the inventors of the present invention teaches a human synthetic peptide-based influenza vaccine comprising at least four epitopes of influenza virus, said influenza virus epitopes being reactive with human cells, said epitopes comprising:
(i) one B-cell haemagglutinin (HA) epitope; (ii) one T-helper haemagglutinin (HA) or nucleoprotein (NP) epitope that can bind to many HLA molecules; and (iii) at least two cytotoxic lymphocyte (CTL) nucleoprotein (NP) or matrix protein (M) epitopes that are restricted to the most prevalent HLA molecules in different human populations, in particular specific ethnic or racial groups. The influenza peptide epitopes can be expressed within recombinant Salmonella flagellin. That vaccine requires the cumbersome preparation of at least four chimeric polypeptides.
PCT International Publication WO 2007/066334 to some of the inventors of the present invention discloses a vaccine able to elicit long term and cross-strain protection comprising a plurality of chimeric proteins comprising at least two influenza virus peptide epitopes wherein at least one epitope is an influenza A virus matrix protein M peptide epitope and the second epitope is a hemagglutinin HA peptide epitope. In this case also the influenza peptide epitopes can be expressed within recombinant Salmonella flagellin.
Mammals often have acquired immune responses to flagellar antigens. However, clinical data have shown that effective doses of recombinant flagellin influenza in animals have adverse effects in human subjects, including high fever, probably due to the high flagellin/antigen ratio. It is also suspected that high concentrations of flagellin have a transient effect on the heart.
Thus there is an unmet need for an influenza peptide epitope-based vaccine which can induce humoral and cellular responses that are long-lasting with broad specificity. There is also a need for a vaccine with simplified production and quality control processes.